Toll-like Receptor 4 (TLR4) recognizes lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. MD-2 is a 160-amino acid glycoprotein that associates with the extracellular domain of TLR4 and is essential for the cellular distribution of TLR4. LPS is the major ligand for the TLR4/MD-2 complex. In addition to LPS, TLR4 can recognize several other ligands such as: taxol, envelope proteins, fusion proteins; and endogenous ligands, such as: oligosaccharides of hyaluronic acid and fibrinogen.? ? Our goal is to determine the high resolution crystal structure of TLR4 and TLR4 in complex with MD-2. As previously reported, the baculovirus expression system was used, that provides correct glycosylation, folding and secretion of the target proteins. The purified hTLR4, hMD-2, mTLR4, mMD2 or hTLR4/hMD-2, mTLR4/mMD2 complexes were used in extensive crystallization trials with a Mosquito crystallization robot, but we were unable to obtain crystals. Co-expression for both human and mouse TLR4/MD-2 was performed, the complex purified and used in crystallization trials without success.? ? We shifted our focus on the TLR4/MD-2 signaling complex. For this, the purified hTLR4/hMD-2 complex was further complexed with ultrapure bacterial LPS or synthetic lipid A derivatives, in the presence of LBP and CD14. After identifying the best ligand, milligram quantities of hTLR4/hMD-2/ligand were generated, further purified and used in crystallization trials. Tiny crystals were obtained with a particular condition, which are being further optimized now.
